Rheumatoid Arthritis Patients On Persistent Moderate Disease Activity On Biologics Have Adverse 5-years Outcome Compared To Persistent Low-remission Status Yet They Represent A Heterogeneous Group: Lower-moderate Has Better Functionality And Fewer Serious Adverse Events Than Higher-moderate Subgroup

Background The long-term outcome of rheumatoid arthritis (RA) patients who in clinical practice, exhibit persistent moderate disease activity (pMDA) despite treatment with biologics has not been adequately studied. Herein, we analyzed the 5-year outcome of the pMDA group and assessed for within-group heterogeneity. Methods We included longitudinally-monitored RA patients from the Hellenic Registry of Biologic Therapies with persistent (cumulative time ≥ 50% of a 5-year period) moderate (pMDA, 3.2<DAS28 ≤ 5.1) or remission/low (pRLDA, DAS28 ≤ 3.2) disease activity. The former was further classied into persistent lower-moderate (plMDA, DAS28<4.2) and higher-moderate (phMDA, DAS28 ≥ 4.2) subgroups. Five-year trajectories of functionality (HAQ) was the primary outcome in comparing pRLDA versus pMDA and assessing heterogeneity within the pMDA subgroups through multivariable mixed-effect regression. Results We identied 295 patients with pMDA and 90 patients with pRLDA, the former group comprising of plMDA (n=133, 45%) and phMDA (n=162, 55%). pMDA was associated with worse 5-year functionality trajectory than pRLDA (+0.27 HAQ units, CI 95% +0.22 to +0.33; p<0.0001), while the phMDA subgroup had worse 5-year functionality than plMDA (+0.26 HAQ units, CI 95% 0.18 to 0.36; p<0.0001). Importantly, higher persistent disease activity was associated with more serious adverse events (SAEs) [pRLDA: 0.2±0.48 vs pMDA: 0.5±0.96, p=0.006; plMDA: 0.32 ±0.6 vs phMDA: 0.64 ±1.16, p=0.038). Male gender (p=0.017), lower baseline DAS28 (p<0.001), HAQ improvement >0.22 (p=0.029) and lower average DAS28 during the rst

although improve disease activity, they still have moderate disease activity (MDA) after treatment with the rst bDMARD (6).
Outcomes of patients with residual MDA while on treatment are available only from trials focusing on early-onset disease treated with conventional synthetic DMARDs (csDMARDs) (9,10) and a pooled analysis of randomized controlled clinical trials of TNFis reported on early (1-year) outcomes (1). Moreover, most of the registries report on disease activity status at prespeci ed time-points after treatment, while data indicative of long-term longitudinal disease activity course (i.e. time-averaged disease activity) are limited. To this end, data for RA patients on bDMARDs having persistently MDA are scant. Rheumatologists in clinical practice often confront with patients who have residual disease activity even after switching biologic disease modifying drugs. Therefore, and given the lack of data for this group, we sought to assess the long-term outcome of RA patients on bDMARDs who exhibit persistent moderate disease activity (pMDA) in clinical practice context.
Herein, we analyzed data from the Hellenic Registry of Biologic Therapies to evaluate the long-term (5years) outcome of RA patients on bDMARDs who exhibit pMDA. We aimed to compare the functional status (HAQ) at 5 years and its longitudinal course (trajectories) in patients with pMDA versus those on persistent lower in ammatory burden, as well as to assess for clinical heterogeneity within pMDA. We also looked for early predictors of patients' classi cation into the distinct persistent disease activity groups.

Data source
The Hellenic Registry of Biologic Therapies (HeRBT) was established in 2004 as a multicenter (7 centers) prospective observational cohort of patients with in ammatory arthritides (6). RA and spondyloarthritis patients are included in the HeRBT at the initiation of rst bDMARD and are followed-up for as long as they receive bDMARDs. Detailed analysis of protocol is given elsewhere (6,11). Treatment decisions (bDMARD selection, co-medication, dosage adjustments/switches) were made at the discretion of treating rheumatologists based on clinical assessments, national guidelines and patient's preferences.
Ethical approval was obtained from the Institutional Review-Board of the University Hospital of Heraklion, Crete (decision-number:1476/20-03-2012) along with participants' informed consents.

Patients
We analyzed all adult patients diagnosed with RA who were registered for the rst time in HeRBT until 31/5/2013 and had continuous follow-up for at least 5 years, irrespective of bDMARD treatment switches. Data were censored when patients completed 5 years of follow-up.
Longitudinal clinical data Data harmonization was performed to summarize longitudinal disease activity (assessed by DAS28 index) and functionality (assessed by Health-Assessment-Questionnaire (HAQ)) assessments in clinically relevant therapy time (TT) intervals. Speci cally, we de ned a total of eight TT intervals spanning sequentially over the 5 years follow-up period, which included six semesterly for the rst 2 years, followed by 3 yearly time-intervals. DAS28 and HAQ averages were computed for each patient, in each TT interval, from all clinical assessments conducted in the speci c time period.
De nitions and clustering by persistent disease activity Patients were classi ed into persistent low (pRLDA) and persistent moderate (pMDA) disease activity groups, if they correspondingly had DAS28≤3.2 and 3.2<DAS28≤5.1 for cumulative time percentage ≥50% of the 5-year follow-up time. Cumulative time percentage (CTP) of a DAS28 range was de ned as the ratio of TT intervals that have DAS28 in the speci c range. A patient was clustered in a persistent disease activity group when a speci c DAS28-range occurred for CTP≥50% or equivalently for at least 4 out of the 8 TT intervals (any of them).
To assess for within-group heterogeneity, patients with pMDA were further sub-divided into persistent lower-moderate (plMDA) and higher-moderate (phMDA) disease activity groups, when they ful lled the additional criterion of having persistent DAS28<4.2 and DAS28≥4.2, respectively for CTP≥50%, as de ned above. Any con icting cases, where patients could be classi ed in two different persistent disease activity groups, were resolved by the preference policy of the worst-case scenario (classi cation in the higher disease activity group).

Statistical analysis
Summary descriptive measures were used on baseline characteristics of the persistent disease activity groups. Non-parametric hypothesis tests (Kruskal-Wallis, Wilcoxon rank-sum, and chi-squared, as appropriate) were used to compare groups' differences at baseline characteristics (Bonferroni-corrected p-value=0.0019 to account for multiple comparisons) and also to compare groups' 5-year outcomes regarding functional status (HAQ) and cumulative serious adverse events (SAEs). To estimate the required sample size to compare functionality, we formulated the null hypothesis that functionality (HAQ) at 5 years would not differ signi cantly between pRLDA and pMDA groups (i.e. difference in HAQ ≤0.22 units). To reject this null hypothesis with 80% power (α=0.05) and assuming 0.5 mean HAQ (0.5 std) in the pMDA and 0.28 in the pRLDA group and patient allocation ratio pRLDA:pMDA to be 1:2, the estimated required sample size was at least 61 and 122 patients in the pRLDA and pMDA groups, respectively.
Multivariable mixed-effect regression analysis was used to associate the persistent disease activity groups pRLDA and pMDA with different 5-year functionality trajectories. Additionally, multivariable mixedeffect regression was performed for pMDA group to assess for clinical heterogeneity (5-year functionality trajectories) between plMDA and phMDA subgroups. In the analyses, HAQ was modeled as the dependent outcome variable while the persistent disease activity group (or subgroup) was modeled as xed-effect variable (categorical, dummy-coded with reference-category the lower persistent disease activity group).
We accounted for individual patient variability with a random effect on the patient level (random intercept for each patient) and adjusted for possible confounding effects of gender, age and disease duration ( xed-effects). Time in treatment course was modeled as xed-effect categorical variable (dummy-coded, 9-values, reference-category baseline, remaining values represent the 8 TT intervals). This time representation was selected to model the change in HAQ in each TT interval compared to baseline.
Alternative representations of groups' interactions with time did not yield signi cantly better results on comparison metrics of AIC, BIC and negative log-likelihood.
Multivariable logistic regression was also performed to analyze for early predictors that classify in pMDA (versus pRLDA) group. The model was adjusted for gender, age (per year), disease duration (per year) and previous csDMARDs at baseline (binary, true for csDMARDs<2). The model also included the following patient's characteristics from baseline and rst therapy semester: baseline disease activity (DAS28), baseline functionality (HAQ), anti-TNF treatment initiation, average disease activity in rst semester (DAS28-average) and functionality difference (ΔHAQ) in rst semester's average (HAQ-average) from baseline (binary, true for difference<-0.22). Predictive e ciency of the model was evaluated using the 10fold cross-validation process (90% training set, 10% test set and 10 repetitions without resubstitution) in the samples of the analysis cohort, in order to avoid over tting and selection bias. The e ciency metrics, accuracy (ACC), sensitivity or true positive rate (TPR), speci city or true negative rate (TNR) and the area under the receiver-operating-characteristic curve (AUC), were averaged from 10 independent datasets extracted from the cross-validation process.

Missing data management
Patients who had proportionally more missing than existing DAS28-assessments, were excluded in order to maintain data quality. Missing DAS28-data in the rest of the patients, were imputed with a multivariable mixed-effect regression model, due to the time-dependent nature of the longitudinal DAS28data (repeated correlated DAS28-measurements in the same patients). The model was adjusted on gender, age and disease duration ( xed-effects) and included a random effect on the patient level and a xed-effect variable for time (as in the aforementioned mixed-effect model). All analyses were performed in MATLAB 9.2 statistical toolbox.

Cohort characteristics
The analysis cohort included 385 patients from the HeRBT registry that ful lled two criteria, (a) they had at least 5 years follow-up and (b) they exhibited persistent low or moderate disease. This cohort was selected out of the 1466 RA patients included in the registry until May 2013, after excluding 763 patients due to < 5 years follow-up, 166 patients having missing longitudinal DAS28-data > 50%, 142 exhibiting persistent high in ammatory burden and 10 patients not exhibiting any persistent disease activity level. This was a cohort mostly with established RA (mean disease duration 9.2 years), 70 patients had disease duration < 2 years and the mean monitoring duration was 7.5 years (Table 1). Patients were treated with an average of 2.34 (± 1.13) csDMARDs prior to rst bDMARD, while 279 (72%) were on combination with methotrexate. At the end of 5 years, 208 (54%) patients received a second and 99 (26%) received a third sequential bDMARD. Persistent disease activity groups characteristics A total 90 (23%) and 295 (77%) patients were categorized in the pRLDA and pMDA groups, respectively. Patients in the pMDA group were further categorized in subgroups plMDA and phMDA including 133 (45%) and 162 (55%) patients, respectively (Table 1). Patients in the pMDA group were older (58 ± 12 vs 52 ± 13 years; p < 0.0019 Bonferroni corrected) and more frequently females (82% vs 58%, p < 0.0019) as compared to pRLDA. At inclusion, patient disease activity (mean DAS28 4.9 ± 1.0 vs 5.9 ± 0.9, p < 0.0019) and functionality (mean HAQ 0.63 ± 0.4 vs 0.9 ± 0.5, p < 0.0019) were higher in pMDA than pRLDA respectively. Analysis within the pMDA group showed that patients in the plMDA subgroup were younger (p = 0.0005) with lower baseline disease activity (mean DAS28 5.6 ± 0.7 vs 6.0 ± 0.9, p < 0.0001) than phMDA patients. A representation of the DAS28 course for each patient in the pRLDA and pMDA groups is provided in Fig. 1 while Fig. 2  Subgroup phMDA was associated with worse 5-year functionality than plMDA Clinical heterogeneity has been reported for the MDA group of patients on csDMARDs while limited data are available concerning bDMARDs. We assessed whether our cohort of RA patients on bDMARDs having persistent moderate disease activity represents a heterogenous group. For this, we compared lower and higher pMDA subgroups, the plMDA and phMDA, respectively. The 5-year functionality trajectories (average HAQ in the 8 TT intervals) of the patients classi ed in plMDA and phMDA subgroups are presented in Fig. 3, revealing a clear distinct trajectory for each subgroup. In multivariable mixed-effect pMDA group was associated with worse 5-year functionality than pRLDA One of the main aims of this study was to investigate whether patients on pMDA have adverse long-term prognosis compared to patients on lower chronic in ammatory burden. The 5-years functionality (HAQ) trajectories (average DAS28 in the 8 TT intervals) of the pRLDA and pMDA groups are presented in Fig. 3, showing a clear distinct trajectory for each group. In multivariable mixed-effect regression analysis (Table 2), the pMDA group was associated with worse 5-year functionality trajectory than the pRLDA group (+ 0.28 higher HAQ trajectory in pMDA, 95% CI + 0.18 to + 0.39, p < 0.0001). Analysis was adjusted for possible confounding effects on gender, age and disease duration. Interestingly, the 5-year functionality was also signi cantly worse in females than males (+ 0.13 higher HAQ trajectory in females, 95% CI + 0.04 to + 0.23, p = 0.008) and in older patients (+ 0.009 higher HAQ trajectory per 1-year, 95% CI + 0.006 to + 0.012, p < 0.0001).  (Table 3), the phMDA subgroup was associated with worse 5-year functionality trajectory than plMDA subgroup (+ 0.26 higher HAQ trajectory in pMDA, 95% CI + 0.17 to + 0.36, p < 0.0001). Analysis was adjusted for possible confounding effects on gender, age and disease duration. The 5-year functionality was also signi cantly worse in females than males (p = 0.04) and in older patients (p < 0.0001). At 5 years, group phMDA had worse functionality status than plMDA (HAQ 0.41 ± 0.38 vs 0.66 ± 0.52 respectively, p < 0.001), although both groups were associated with improved functionality as compared to baseline (HAQ decrease from baseline: -0.38 HAQ, 95% CI -0.463 to -0.297, p < 0.0001). The differentiation of 5-year functionality between subgroups plMDA and phMDA indicates heterogeneity within the pMDA group. We also assessed SAEs occurrence during the course of the follow-up to estimate for any differences in the long-term outcome between distinct patients groups. The 5-year cumulative SAEs trajectories of pRLDA and pMDA groups as well as of the plMDA and phMDA subgroups are presented in Fig. 4, showing a clear distinct trajectory for each group and subgroup respectively. At 5 years, pMDA group had higher occurrence of SAEs than pRLDA group (0.2 ± 0.48 in pRLDA vs 0.5 ± 0.96 in pMDA, p = 0.006). In addition, the phMDA subgroup had also higher occurrence of SAEs than the phMDA subgroup (0.32 ± 0.6 in vs 0.64 ± 1.16 respectively, p = 0.038). The differentiation between subgroups plMDA and phMDA in serious adverse events occurrence, further supports the heterogeneity within the pMDA group.
Early predictors for classi cation between pRLDA and pMDA groups In view of the aforementioned clinically meaningful differences between pRLDA and pMDA patient groups, we developed a predictive model for the early classi cation of patients using data from patients' early therapy months ( rst semester of treatment). Multivariable logistic regression analysis (

Discussion
We characterized the long-term prognosis of RA patients with persistent moderate disease activity (MDA) under bDMARDs treatment in the context of clinical practice. We found that a substantial proportion of patients although improved disease activity status and function, still manifest pMDA irrespective of treatment modi cations. Most importantly, pMDA was associated with worse long-term (5-year) outcomes (functional limitation and serious adverse events) than persistent lower in ammatory burden. Interestingly, the subgroup with persistently lower-MDA had better long-term outcomes than those with persistently higher-MDA.
An important nding of this study was that persistent MDA was linked to signi cantly worse functionality trajectory and higher occurrence of serious adverse events during 5 years of bDMARDs therapy, as compared to persistent low disease activity or remission ( Fig. 3 and Table 2). Interestingly, residual disease activity was the major contributor to HAQ increase over time (Table 2). These data are comparable to those from early RA cohorts on csDMARDs assessing the cumulative effect of disease activity on RA-related outcomes (9,10,12). Although one might argue that this nding is an expected one, we considered clinically important and novel to focus for the rst time on patients treated with bDMARDs, which might exert differential immunomodulatory effects as compared to csDMARDs. For example, it has been shown that both TNFi and tocilizumab may inhibit joint destruction effectively even when residual disease activity exists, which is not the case for methotrexate (13)(14)(15).
Another key nding of our study is that MDA patients comprise a heterogeneous group in terms of outcome. Patients with persistent lower-MDA (plMDA) have signi cantly better 5-year functionality trajectory than those in persistent higher-MDA (phMDA). This agrees with studies focusing on early-onset RA and csDMARDs treatments, showing that patients on the lower end of MDA have signi cantly better outcomes than those on the higher end (10,16). Notwithstanding the fact that observational studies cannot provide direct support for management strategies, we consider our results to be of clinical importance and relevant to the T2T concept. Firstly, our data provide further support to the validity of T2T in clinical practice, since there was a clear superiority for all long-term outcomes in the persistent remission/low as compared to persistent moderate disease activity group. Moreover, the heterogeneity of outcomes in lower and higher MDA patients can assist T2T strategies to tailor treatments for these subgroups in order to improve outcomes.
Long-term prognosis of RA largely depends on the disease in ammatory burden and associated comorbidities. One of the limitations in the literature, is that the majority of short-and even long-term studies evaluate RA-related in ammation cross-sectionally (single time-point). However, values representative of longitudinal course of disease activity and its effect over time are considered to provide more valuable information. One such approach is the average disease activity from multiple years of treatment (10), while another is the area under the curve of DAS28 course which was associated with both radiographic progression (17) and the risk for cardiovascular diseases (CVD) (18,19). In the present study, we applied the cumulative time percentage (CTP) that DAS28 falls within a speci ed range during follow-up (CTP of DAS28-range), as indicative of long-term longitudinal disease activity course. We consider our approach more appropriate for patient group classi cation than using AUC of DAS28-course for the following reasons. Firstly, compared to the AUC, the CTP is interpretable and exible enough to support more restricted or loose speci cations of MDA (different CTP thresholds). Secondly, it imposes a constraint for a proportion of the treatment course (≥ 50%) to exhibit moderate disease activity while it intelligently ignores possible uctuations in the rest of the proportion. Finally, and most importantly, the AUC method may not be able to distinguish a persistent moderate disease activity trajectory from one uctuating equally between low and high disease activity levels, since these trajectories may exhibit approximately equivalent AUC values.
Studies analyzing persistent disease activity status from cohorts of biologics have focused on persistent remission (20)(21)(22)(23)(24)(25)(26). Herein we focused on persistent residual disease activity (pMDA), and we found that from patients in persistent moderate or lower in ammatory burden treated on bDMARDs, 23% were classi ed in a persistent low or remission status while 77% still exhibited substantial in ammatory burden (pMDA) after 5 years of therapy. Even though this seems as a rather high number, yet available data from registries have shown that only 8.2-21% of bDMARDs treated patients are classi ed as being in persistent remission (20)(21)(22)(23)25). Of note, pMDA patients in our cohort differ from pRLDA even from baseline and could be divided further in two heterogenous subgroups. Multivariable analysis revealed gender, lower disease activity (DAS28) at baseline and at rst semester's average and functionality improvement (greater than 0.22 HAQ units) in rst semester's average compared to baseline, as signi cant predictors for early classi cation in persistent low compared to moderate disease activity status ( Table 4).
One of the limitations of this study is patients' missing follow-up data. In order to address missing data and also maintain data quality, we excluded patients with large percentage of missing DAS28-data (> 50%) and imputed missing DAS28-data in the rest of the patients. Of note, the imputed-dataset (385 patients) compared to the non-imputed (292 patients) included additionally 93 (24%) patients, and results were similar in both datasets (groups pRLDA and pMDA differentiated in functionality trajectory and SAEs, data not shown).
Another limitation of this study can be considered the merging of remission and low disease activity groups. The pRLDA group included 52 patients in persistent remission, 20 in persistent low disease activity and 18 with uctuations between low and remission disease activity. Future studies in larger datasets could focus in persistent strictly-low (2.6 ≤ DAS28 ≤ 3.2) and persistent moderate (3.2 < DAS28 ≤ 5.1) disease activity comparison.
In order to evaluate the robustness of the methodological approach, we performed sensitivity analysis in shorter (3-year) therapy duration with similar results (groups pRLDA and pMDA differentiated in functionality trajectory). Furthermore, analysis on groups' differences regarding patients' inclusion-year

Conclusions
Our analysis revealed that a considerable proportion of RA patients on bDMARDs in clinical practice, although improve disease activity status, still manifest persistent moderate disease activity. This state was associated with adverse 5-year outcomes and was also found to present internal heterogeneity, while predictors were analyzed to assist for early patient classi cation. These ndings further support the value of T2T strategy in order to improve long-term outcome and highlight the need for further targeted studies on persistent MDA state and its heterogenous subgroups. HAQ Difference; ACC: Accuracy; TPR: Sensitivity or True Positive Rate; TNR: Speci city or True Negative Rate; AUC: Area Under the Receiver-operating-characteristic Curve; CV: Cross-validation; T2T: Treat to Target.

Declarations
Ethics approval and consent to participate Ethical approval was obtained from the Institutional Review-Board of the University Hospital of Heraklion, Crete (decision-number:1476/20-03-2012). Participants provided informed consents.

Consent for publication
Not applicable.

Availability of data and materials
The data and analytic methods that support the ndings of this study are available to quali ed investigators by request to the corresponding author.   DAS28 trajectories of patient groups pRLDA (remission-low), pMDA (moderate) and subgroups plMDA (lower-moderate), phMDA (higher-moderate). RA: Rheumatoid Arthritis; DAS28: Disease activity score with 28 joint counts; pRLDA: Patients with persistent remission or low disease activity; pMDA: Patients with persistent moderate disease activity; plMDA: Patients with persistent lower moderate disease activity; phMDA: Patients with persistent higher moderate disease activity.